Plasmid DNA is an important product for gene therapy applications. It is grown in host cells that are then lysed to release the plasmid DNA. The lysate is then clarified and the plasmid DNA is recovered using various regimes such as chromatography and the like.
Methods for clarifying lysate streams containing plasmid DNA have been described in WO 00/05358 and WO 97/23601. In both, the lysate is subjected to a centrifugation step that separates the plasmid DNA and smaller solids and particulates from the larger solids such as cell wall debris and the like. The supernatant containing the plasmid DNA is then carefully removed from the centrifuge and subjected to a series of filtration steps comprised of a coarse filter formed of a series of filter cloths (20 microns average pore size), followed by a prefiltration step (1-2 microns) and a final filtration step (about 0.2 microns).
Others have suggested using pad filters such as cellulosic fibers containing various fillers such as diatomaceous earth, clays, etc. or using a tangential flow filtration system (TFF) to clarify the lysate stream after centrifugation.
All of these systems are less than optimal for the recovery of plasmid DNA. Plasmid DNA typically accounts for less than 1%, more typically less than 0.5% by weight of the lysate stream, making the need for high recovery of the plasmid DNA important.
Centrifugation applies a shear to the constituents of the lysate stream which can damage the plasmid DNA. Additionally, recovery of the plasmid DNA containing portion of the stream is subjective and either is less than the total amount available or includes additional solids and particulates that adversely affect the downstream filtration steps (typically by prematurely clogging the filters.). Moreover, centrifugation requires an investment in capital equipment which is expensive, difficult to sanitize to FDA standards and requires the system be run in a batch format.
The proposed filtration train of the prior art has multiple steps and has a relatively low yield of plasmid DNA, typically less than about 80%. Under optimal laboratory conditions, these systems have yielded less than 90% of the available plasmid DNA. This is due to several factors. The selected filtration elements tend to interact with the plasmid DNA, binding some of it to their system. Additionally, each system has a minimum holdup volume that retains some of the plasmid DNA and never passes it on to the next stage of filtration.
Filter pads have not been an acceptable alternative as they irreversibly absorb a relatively high level of plasmid DNA, leading to low yields.
The use of TFF has been minimal as the TFF system with its constant washing of the plasmid DNA across the face of the membrane and its recirculation through one or more pumps applies a high shear on the plasmid DNA leading to reduced yields and reduced efficacy of the plasmid DNA that is recovered. Additionally, the yield of TFF devices is adversely affected by the high hold up volume of the TFF devices.
What is needed is a system and method of clarification that reduces the loss of yield and maintains the efficacy of the plasmid DNA recovered.